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D A Eisner

Publications and source records attributed to D A Eisner.

At least 19 recordsLinked to original sources
PubMed1999

The role of sarcolemmal Ca2+-ATPase in the regulation of resting calcium concentration in rat ventricular myocytes.

1. The aim of this work was to investigate the role of sarcolemmal Ca2+-ATPase in rat ventricular myocytes. We have measured intracellular Ca2+ concentration ([Ca2+]i) using indo-1. The actions of the ATPase inhibitor carboxyeosin were studied. 2. Carboxyeosin increased resting [Ca2+]i and the magnitude of the systolic Ca2+ transient and slowed the rate of its relaxation by 5 %. 3. Carboxyeosin increased the magnitude of the caffeine-evoked increase in [Ca2+]i and slowed its relaxation by 20 %. Removal of extracellular Na+ slowed the rate constant by 80 %. When Na+ was removed in a carboxyeosin-treated cell, the caffeine-evoked increase in [Ca2+]i did not decay. 4. Carboxyeosin increased the integral of the Na+-Ca2+ exchange current activated by caffeine. This is, in part, due to an increase in sarcoplasmic reticulum Ca2+ content. 5. When extracellular Na+ was removed, there was a transient increase in [Ca2+]i which then decayed. The rate of this decay was slowed by carboxyeosin by a factor of about four. The residual decay could be abolished with caffeine. 6. In the absence of extracellular Na+, increasing extracellular Ca2+ concentration ([Ca2+]o) elevated [Ca2+]i. In carboxyeosin-treated cells, [Ca2+]i was much more sensitive to [Ca2+]o. 7. These results demonstrate the role of a carboxyeosin-sensitive Ca2+-ATPase in the control of resting [Ca2+]i and the reduction in [Ca2+]i following an increase in [Ca2+]i.

Animals↗
PubMed1999

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes.

This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca2+]i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca2+]i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114+/-18 micromol l-1 and Kd of 0.59+/-0.17 microM (n=8). During the systolic rise of [Ca2+]i, the measured changes of [Ca2+]i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca.

Animals↗
PubMed1998

Properties of voltage-activated [Ca2+]i transients in single smooth muscle cells isolated from pregnant rat uterus.

1. The intracellular calcium concentration ([Ca2+]i) was measured at 35 degrees C using the fluorescent indicator indo-1 in patch-clamped, single uterine myocytes from pregnant rats to investigate the relationship between depolarization, Ca2+ current (ICa) and [Ca2+]i. 2. Membrane depolarization activated ICa and produced a [Ca2+]i transient. The rapid increase in [Ca2+]i occurred at the same time as the inward ICa. Both ICa and the increase in [Ca2+]i were abolished by nifedipine (10 microM). 3. When the membrane potential was held at -80 mV the threshold depolarization for an increase in [Ca2+]i was about -55 to -50 mV. As the magnitude of the depolarization was increased to about 0 mV there was an increase in the size of both ICa and the increase in [Ca2+]i. As the magnitude of the depolarization was further increased both ICa and the [Ca2+]i increase declined. 4. When the depolarizing pulses were applied at 3 Hz to mimic normal action potentials then the individual [Ca2+]i transients did not fully relax and a tetanic rise of [Ca2+]i was observed. Under these conditions, there was not a simple relationship between the magnitude of the Ca2+ response and Ca2+ entry. When pairs of depolarizing pulses were applied, the increase in [Ca2+]i produced by the second pulse was larger (in relation to the magnitude of the L-type Ca2+ current) than that produced by the first pulse. This facilitation was abolished by both ryanodine and cyclopiazonic acid suggesting a role for release from intracellular stores. 5. We conclude that the L-type Ca2+ current is the major source of Ca2+ ions entering the cell to produce the [Ca2+]i transient on depolarization. The magnitude of the increase in [Ca2+]i may, however, be amplified by Ca2+-induced Ca2+ release.

Animals↗
PubMed1998

The effect of tetracaine on stimulated contractions, sarcoplasmic reticulum Ca2+ content and membrane current in isolated rat ventricular myocytes.

1. The effects of tetracaine were examined on rat ventricular myocytes. In both field-stimulated and voltage-clamped cells tetracaine (100-200 microM) produced an initial decrease of contraction before a recovery towards the control level. Removal of tetracaine produced a transient overshoot of contraction to levels greater than the control. 2. The transient decrease of contraction produced by tetracaine was accompanied by a small transient increase in the integral of the L-type Ca2+ current and a larger transient decrease of the Na+-Ca2+ exchange current on repolarization. These are attributed to decreased systolic release of Ca2+. On removal of tetracaine there was an increase of the Na+-Ca2+ exchange current. Before the addition of tetracaine, calculated Ca2+ influx and efflux across the sarcolemma were approximately equal. On adding tetracaine, efflux was transiently less than influx and, on removal of tetracaine, efflux was greater than influx. 3. These changes in Ca2+ fluxes result in an increase of cell Ca2+ during exposure to tetracaine. The calculated magnitude of this increase was equal to that measured directly by applying caffeine (20 mM) to release sarcoplasmic reticulum (SR) Ca2+ and integrating the resulting Na+-Ca2+ exchange current. 4. It is concluded that the effects of tetracaine can be accounted for by depression of calcium-induced Ca2+ release (CICR). The response is transient because the inhibition is compensated for by an increase of SR Ca2+ content such that there is no steady-state effect on the magnitude of the systolic Ca2+ transient. The consequences of this result for the effects of other modulators of CICR are discussed.

Animals↗
PubMed1998

The effects of changing intracellular pH on calcium and potassium currents in smooth muscle cells from the guinea-pig ureter.

Guinea-pig ureteric smooth muscle is unusual in that intracellular acidification increases and alkalinization decreases force production. To help elucidate the mechanism underlying these effects on force we have investigated the effects of changing intracellular pH on both calcium and potassium currents in single cells isolated from the guinea-pig ureter to determine their possible role in force development. Depolarization to +40 mV resulted in a fast transient outward current which was inhibited by 4-aminopyridine but not tetraethylammonium. Intracellular alkalinization (20 mM trimethylamine) increased this current to 179 +/- 24% of the control and resulted in the development of a slowly activating large outward current which was inhibited by tetraethylammonium and washout. Acidification (40 mM sodium butyrate) decreased the fast transient outward current to 58 +/- 3% of the control and did not produce a slowly activating current. When potassium was replaced by caesium in the pipette solution, depolarization to 0 mV resulted in an inward calcium current which was abolished by nifedipine. Intracellular alkalinization increased this current to 126 +/- 11% of the control whereas acidification had the opposite effect, decreasing it to 55 +/- 10%. Furthermore, current-clamp experiments showed that intracellular alkalinization inhibited the amplitude of the action potential, therefore decreasing excitability of the cell. From our results, we suggest that the predominant effects of intracellular pH on force production in the guinea-pig ureter are mediated via the modulation of outward potassium currents (thereby reducing excitability of the tissue) rather than the effects on the inward calcium current.

Animals↗
PubMed1998

Ca-activated chloride current and Na-Ca exchange have different timecourses during sarcoplasmic reticulum Ca release in ferret ventricular myocytes.

The aim of this work was to measure membrane currents activated by Ca release from the cardiac sarcoplasmic reticulum (s.r.). Intracellular Ca concentration ([Ca2+]i) was measured using fluo-3 in patch clamped cells. Calcium release from the s.r. (whether occurring spontaneously or evoked by caffeine) produced changes of membrane current which could be separated into a Ca-activated Cl current which was inhibited by DIDS or Cl removal and a Na-Ca exchange current. Both these currents had different time courses from the measured [Ca2+]i. Furthermore the Ca-activated Cl current decayed more quickly than did Na-Ca exchange. Possible explanations for the different kinetics of these two Ca-sensitive currents are discussed.

Animals↗
PubMed1998

2,3-Butanedione monoxime (BDM) decreases sarcoplasmic reticulum Ca content by stimulating Ca release in isolated rat ventricular myocytes.

The effects of 2,3-butanedione monoxime (BDM) were examined using rat ventricular myocytes loaded with Indo-1 to measure the intracellular Ca concentration ([Ca2+]i). BDM (10 mM) produced a transient increase of the systolic Ca transient with no steady-state effect on its magnitude. This transient increase was more marked when BDM was applied after having decreased the external Ca concentration from 1 to 0.1 mM. There was a transient increase of resting [Ca2+]i in both quiescent and electrically stimulated cells. Prior application of BDM decreased the rise of [Ca2+]i produced by caffeine. In voltage-clamped cells the rise of [Ca2+]i produced by BDM was accompanied by a transient inward current attributed to the electrogenic Na-Ca exchange. The amount of Ca lost from the cell upon application of 10 mM BDM could be estimated either from the integral of the BDM-evoked current or from the reduction of the integral of a caffeine-evoked current and corresponded to about 50% of the sarcoplasmic reticulum (s.r.) Ca content. The decrease of s.r. Ca content and the transient potentiation of the systolic Ca transient suggest that BDM acts by stimulating Ca-induced Ca release. These effects must be allowed for when using BDM.

Animals↗
PubMed1998

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats.

In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca2+]i) is determined by Ca2+ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca2+ pump inhibitor, carboxyeosin, on the rate of the [Ca2+]i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp technique to measure [Ca2+]i simultaneously with transmembrane calcium current (ICa). [Ca2+]i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity. The rate of [Ca2+]i removal was calculated from the falling phase of the [Ca2+]i transient. Pre-treatment of the cells with 2 microM carboxyeosin led to a marked decrease in the rate of [Ca2+]i transient decay, suggesting that the sarcolemmal Ca2+ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca2+]i decay, indicating an important role for the Na+/Ca2+ exchange. When both the sarcolemmal Ca2+ pump and Na+/Ca2+ exchange were inhibited the cell failed to restore [Ca2+]i after the stimulation. Comparison of the rate constants of [Ca2+]i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca2+]i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na+/Ca2+ exchanger and the intracellular calcium stores.

Animals↗
PubMed1998

Stimulation of Ca-induced Ca release only transiently increases the systolic Ca transient: measurements of Ca fluxes and sarcoplasmic reticulum Ca.

OBJECTIVE: To investigate the effects of stimulating calcium induced Ca release with low concentrations (100-200 microM) of caffeine and, in particular, to study the cellular mechanisms responsible for the transient responses found previously. METHODS: Experiments were performed on isolated rat ventricular myocytes. Intracellular calcium concentration ([Ca2+]i) was measured with Indo-1, the cells were voltage-clamped with the perforated patch technique and sarcoplasmic reticulum (s.r.) Ca content was estimated from the integral of the caffeine-evoked current. RESULTS: The systolic Ca transient produced by the first depolarization in the presence of caffeine was larger than the control. Over the next few pulses the magnitude of the Ca transient returned to control levels despite the maintained presence of caffeine. The s.r. Ca content was decreased by 9% after one pulse in caffeine and by 21% after several pulses in caffeine. The first pulse in the low concentration of caffeine was followed by an enhanced inward (Na-Ca exchange) current tail indicating increased efflux of calcium from the cell. The extra loss of calcium calculated from the tail current agreed quantitatively with that from the change of s.r. Ca content. CONCLUSIONS: These results show that stimulating calcium induced calcium release produces only a transient increase of the systolic Ca transient. This is due to the larger Ca transient decreasing the s.r. Ca content. It is concluded that any agent whose sole mode of action is stimulation of calcium-induced calcium release will not produce a maintained inotropic effect. The consequences of this for the effects of other modulators of calcium induced calcium release are discussed.

Animals↗
PubMed1998

The control of Ca release from the cardiac sarcoplasmic reticulum: regulation versus autoregulation.

This review discusses the mechanism and regulation of Ca release from the cardiac sarcoplasmic reticulum. Ca is released through the Ca release channel or ryanodine receptor (RyR) by the process of calcium-induced Ca release (CICR). The trigger for this release is the L-type Ca current with a small contribution from Ca entry on the Na-Ca exchange. Recent work has shown that CICR is controlled at the level of small, local domains consisting of one or a small number of L-type Ca channels and associated RyRs. Ca efflux from the s.r. in one such unit is seen as a 'spark' and the properties of these sparks produce controlled Ca release from the s.r. A major factor controlling the amount of Ca released from the s.r. and therefore the magnitude of the systolic Ca transient is its Ca content. The Ca content depends on both the properties of the s.r. and the cytoplasmic Ca concentration. Changes of s.r. Ca content and the Ca released affect the sarcolemmal Ca and Na-Ca exchange currents and this acts to control cell Ca loading and the s.r. Ca content. The opening probability of the RyR can be regulated by various physiological mediators as well as pharmacological compounds. However, it is shown that, due to compensatory changes of s.r. Ca, modifiers of the RyR only produce transient effects on systolic Ca. We conclude that, although the RyR can be regulated, of much greater importance to the control of Ca efflux from the s.r. are effects due to changes of s.r. Ca content.

Animals↗
PubMed1997

The role of the sarcolemmal Ca(2+)-ATPase in the pH transients associated with contraction in rat smooth muscle.

1. We have investigated the origin of the intracellular acid pH transients that accompany myometrial contraction. Intra- and extracellular pH were measured with SNARF and intracellular Ca2+ concentration ([Ca2+]i) with indo-1. 2. An intracellular acidification accompanied spontaneous contractions and those elicited by KCl depolarization or the addition of the agonists carbachol or prostaglandin F2 alpha. The size of the acidification increased with the magnitude of the contraction. 3. The intracellular acidification was accompanied by an extracellular alkalinization, showing that it results from proton movement across the surface membrane. Furthermore, it was decreased either by addition of Cd2+ (20 nM, an inhibitor of the sarcolemmal Ca(2+)-ATPase) or by elevating [Ca2+]o. 4. Extracellular alkalinization increased the magnitude of the rise of [Ca2+]i and force produced by KCl. 5. An intracellular acidification was also associated with contraction in the portal vein and ureter. 6. We conclude that the sarcolemmal Ca(2+)-ATPase produces a significant intracellular acidification while removing Ca2+. Both the acidification and decrease of [Ca2+]i will promote relaxation. Since Ca2+ and protons have opposite effects on many cellular processes, this dual regulation by these two ions may be of general importance.

Animals↗
PubMed1997

The effect of tetracaine on spontaneous Ca2+ release and sarcoplasmic reticulum calcium content in rat ventricular myocytes.

1. The effects of tetracaine were studied on voltage-clamped rat ventricular myocytes, which exhibited Ca2+ overload as identified by spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) as shown by the associated contractions. This Ca2+ release was initially abolished by tetracaine before returning at a lower frequency, but greater amplitude, than the control. On removal of tetracaine, there was a burst of spontaneous Ca2+ release activity. All these effects were dose dependent, from 25 to 200 microM tetracaine. 2. The spontaneous Ca2+ release activated an inward Na(+)-Ca2+ exchange current as Ca2+ was pumped out of the cell. The integral of this current (i.e. the Ca2+ efflux) was increased in the presence of tetracaine. The calcium efflux per unit time was unaffected by tetracaine. 3. The SR Ca2+ content was increased by tetracaine, as shown by the integral of the caffeine-evoked Na(+)-Ca2+ exchange current. The increase of SR Ca2+ content was equal to the extra Ca2+ lost from the cell during the burst on removal of tetracaine, and to estimates of the extra calcium gained over the quiescent period following addition of tetracaine. 4. It is concluded that partial inhibition of calcium-induced calcium release increases SR Ca2+ content. In the steady state, cell Ca2+ balance is maintained as the lower frequency of spontaneous release (that activates efflux) is compensated for by their greater size.

Anesthetics, Local↗
PubMed1997

Measurement of sarcoplasmic reticulum Ca2+ content and sarcolemmal Ca2+ fluxes in isolated rat ventricular myocytes during spontaneous Ca2+ release.

1. Intracellular calcium concentration ([Ca2+]i) and Na(+)-Ca2+ exchange currents were measured in calcium-overloaded voltage-clamped rat ventricular myocytes loaded with the Ca(2+)-sensitive fluorescent indicator indo-1. Sarcoplasmic reticulum (SR) Ca2+ content was measured from the integral of the caffeine-evoked current. In cells that had spontaneous SR Ca2+ release in 1 mM external Ca2+ concentration ([Ca2+]o)i raising [Ca2+]o increased the frequency of release with no effect on SR Ca2+ content. In quiescent cells, increased [Ca2+]o produced spontaneous Ca2+ release associated with increased SR Ca2+ content. Further increase of [Ca2+]o had no effect on SR Ca2+ content. The amount of Ca2+ leaving the cell during each release was constant over a wide range of frequencies and [Ca2+]o values. It appears there is a maximum level of SR Ca2+ content, perhaps because spontaneous Ca2+ release results when the content reaches a threshold. 2. From the relationship between [Ca2+]i and Na(+)-Ca2+ exchange current during a caffeine response, it is possible to estimate the changes in Na(+)-Ca2+ exchange current expected from a change of [Ca2+]i. The data show that the calcium oscillations contribute a significant fraction of the total extra Ca2+ efflux induced by increasing [Ca2+]o. Raising [Ca2+]o decreased the rate of calcium removal from the cell as measured from the rate of decay of the caffeine response, suggesting that both inhibition of Ca2+ efflux and increased Ca2+ entry account for the Ca2+ overload at elevated [Ca2+]o. 3. Inhibiting spontaneous SR Ca2+ release increases resting [Ca2+]i. The Ca2+ efflux is identical to that in the presence of release. It is concluded that spontaneous release of calcium, although potentially arrhythmogenic, is an effective way to activate Ca2+ efflux in overloaded conditions and minimizes any increase of diastolic tension.

Animals↗
PubMed1997

A measurable reduction of s.r. Ca content follows spontaneous Ca release in rat ventricular myocytes.

The Ca content of the sarcoplasmic reticulum (s.r.) was measured in voltage-clamped rat ventricular myocytes from the integral of the Na-Ca exchange current evoked by applying caffeine to release the s. r. Ca content. Following spontaneous release of Ca from the s.r., the s.r. Ca content was decreased. The magnitude of this decrease was equal to that of the amount of calcium directly measured to have been pumped out of the cell during the spontaneous release. Following a spontaneous release, the s.r. Ca content recovered linearly. These results are shown to be consistent with the hypothesis that the frequency of spontaneous release is determined by the time taken for the cell and s.r. to reaccumulate the Ca2+ ions pumped out of the cell during spontaneous release.

Animals↗
PubMed1997

Enhanced Ca2+ current and decreased Ca2+ efflux restore sarcoplasmic reticulum Ca2+ content after depletion.

[Ca2+]i was measured using the fluorescent indicator indo 1 in voltage-clamped ferret and rat ventricular myocytes. The Ca2+ content of the sarcoplasmic reticulum (SR) was estimated from the integral of the Na(+)-Ca2+ exchange current activated by caffeine. Refilling of the SR after caffeine removal was enhanced by stimulation. As the systolic Ca2+ transient recovered, the integral of the L-type Ca2+ current decreased and that of the Na(+)-Ca2+ exchange tail current increased. For the early pulses, the gain of Ca2+ via the Ca2+ current is greater than the loss via the exchanger, and during steady state stimulation, the fluxes are equal. The difference in the integrals gives a measure of the net gain of cell Ca2+ with each pulse. When these are summed, the calculated gain of cell Ca2+ agrees well with the increase of SR Ca2+ produced by stimulation, as measured from the caffeine-evoked currents. There was a nonlinear relationship between SR Ca2+ content and the magnitude of the systolic Ca2+ transient such that at high SR Ca2+ content a given increase of content had a greater effect on the Ca2+ transient than did an increase at low SR content. In conclusion, the effects of systolic Ca2+ on the Ca2+ current and Na(+)-Ca2+ exchange current provide a means to regulate SR Ca2+ content and thence the systolic Ca2+ transient.

Animals↗